Cloned leptospira outer membrane protein

ABSTRACT

An antigenic preparation is provided which contains a 63 Kd outer membrane protein from Leptospira which can be used immunologically as a vaccine for leptospirosis caused by this organism. Also provided in the invention are polynucleotides encoding the protein and antibodies which bind the protein which are useful in the diagnosis of leptospirosis.

[0001] This invention was made with Government support by the Veteran'sAdministration Research Advisory Group and Grant Nos. Al-21352,Al-29733, and Al-12601 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to an antigenic preparation andspecifically to a Leptospira outer membrane protein (OmpL2) which isused to induce a protective immune response in animals. Such a proteincan be used immunologically as a vaccine for leptospirosis caused bythis organism. Alternatively, diagnosis of leptospirosis can beperformed by detecting the presence of the protein, antibody to theprotein, or polynucleotide which encodes the protein.

[0004] 2. Description of Related Art

[0005] Leptospirosis is a widespread zoonotic disease caused bypathogenic strains of Leptospira which are capable of infecting mostmammalian species. At present, there are six pathogenic species andthree nonpathogenic species within the genus Leptospira. Infectionoccurs either through direct contact with an infected animal or indirectcontact with contaminated soil or water. In livestock, the diseasecauses economic losses due to abortion, stillbirth, infertility,decreased milk production, and death.

[0006] Efforts to control leptospirosis have been hampered becausevirulent leptospires have the capacity for both long-term survival inthe environment as well as persistent infection and shedding by wildlifeand livestock. Currently available leptospiral vaccines produceshort-term immunity and do not provide cross-protection against many ofthe 170 serovars of pathogenic Leptospira (Thiermann, et al.,J.Am.Vet.Med.Assoc. 184:722, 1984). These vaccines consist ofinactivated whole organisms or outer envelope preparations which produceseroreactivity as determined by microscopic agglutination of intactorganisms. The nature of the protective immunogens in these vaccinepreparations has not been conclusively elucidated, although severallines of evidence suggest that lipopolysaccharide-like substance (LLS)may confer a degree of protection.

[0007] The pathogenesis of leptospirosis is very similar to that ofother spirochetal diseases, including syphilis (caused by Treponemapallidum) and Lyme borreliosis (caused by Borrelia burgdorferi). Bothsyphilis and Lyme borreliosis are characterized by widespreaddissemination early in the course of disease, including invasion of thecentral nervous system. Leptospira share this ability with otherpathogenic spirochetes such that meningitis is a common manifestation ofleptospirosis. Another feature of spirochetal infections is the abilityto persist chronically in the host, as manifested in cases of tertiarysyphilis and chronic Lyme arthritis.

[0008] In attempting to identify leptospiral outer membrane proteins(OMPs), previous research was unsuccessful due to such problems as: 1)the techniques used to identify surface-exposed proteins probablyinvolved damage to the fragile leptospiral outer membrane resulting inexposure of subsurface structures; 2) putative surface-exposed proteinsthat were identified included a 35-36 kD doublet corresponding toLeptospira endoflagella (Kelson, et al., J. Med. Microbiol. 26:47,1988), which are subsurface structures in spirochetes; and 3) use of SDSwhich nonselectively solubilizes proteins irrespective of their nativecellular location.

[0009] Nunes-Edwards, et al. (Infect. Immun. 48:492, 1985) introducedthe use of radioimmunoprecipitation and cell fractionation schemes basedon the use of SDS in an effort to identify leptospiral OMPs. Theleptospires used in their radioimmunoprecipitation procedure weresubjected to high speed centrifugation (20,000×g) prior to the additionof antibody. Such high centrifugal forces cause mechanical disruption ofthe leptospiral outer membrane. Niikura, et al. (Zbl. Bakt. Hyg. A.266:453, 1987) immunoprecipitated SDS-solubilized extracts of virulentand avirulent strains of L. interrogans serovar copenhageni that hadbeen labeled by lactoperoxidase-catalyzed surface radioiodination. Sinceboth of these studies precipitated a 35-36 kD doublet consistent withleptospiral endoflagella, there was a concern as to whether the otherproteins identified might also have a subsurface rather than a surfacelocation.

[0010] Jost, et al. (J. Med. Microbiol. 27:143) characterized amonoclonal antibody with specificity for a 35 kD proteinase K sensitiveantigen which was present in a leptospiral outer envelope preparation.However, to demonstrate binding of the monoclonal antibody byimmunoelectron microscopy, the leptospiral outer membrane had to bedisrupted. Doherty, et al. (J. Med. Microbiol. 28:143) cloned twoleptospiral proteins represented in an SDS-generated outer membranepreparation of L. interrogans, but did not provide corroboratingevidence that these proteins are either constituents of the outermembrane or are surface-exposed.

[0011] Unsuccessful research on the identification of Leptospira and T.pallidum OMPs has shown the importance of taking into accountspirochetal outer membrane fragility and the lack of outer membraneselectivity of ionic detergents such as sodium dodecyl sulfate (SDS)(Cunningham, et al, J.Bacteriol. 170:5789, 1988; Penn, et al., J. Gen.Microbiol. 131:2349, 1985; Stamm, et al., Infect. Immun. 55:2255, 1987).Outer membrane proteins are of great importance because they play a keyrole in bacterial pathogenesis. The identification of outer membraneproteins involved in Leptospira pathogenesis is significant tounderstanding not only leptospiral outer membrane proteins and theirinvolvement in pathogenesis, but also to understanding other spirochetalouter membrane proteins and their role in pathogenesis.

SUMMARY OF THE INVENTION

[0012] The present invention is based on the identification of OmpL2 asa leptospiral outer membrane protein which is associated with pathogenicstrains of Leptospira. Due to spirochetal outer membrane fragility andthe fact that outer membrane proteins are present in small amounts,there have been no definitive reports of membrane spanning spirochetalouter membrane proteins until the present invention. The inventiondescribes a 63 kD outer membrane protein from Leptospira and the geneencoding the protein. The deduced amino acid sequence has a typicalleader peptidase I cleavage site, implying export beyond the innermembrane. The 63 kD protein has been designated OmpL2 for outer membraneprotein of Leptospira. This immunogenic polypeptide is useful forinducing an immune response to pathogenic Leptospira as well asproviding a diagnostic target for leptospirosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows the DNA sequence and deduced amino acid sequence ofOmpL2.

[0014]FIG. 2 shows an amino acid comparison between OmpL2 and eightTonB-dependent outer membrane proteins for seven regions of homologyidentified by Kadner, R., (Molecular Microbiology, 4:2027, 1990).

[0015]FIG. 3 shows a topological model of OmpL2. Membrane spanningbeta-sheets are shown within rectangles in a staggered array with thehydrophobic, membrane-facing residues on the right side of the array.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides an isolated immunogenicpolypeptide from an outer membrane protein of a pathogenic Leptospiraspecies. Also included is a polynucleotide sequence which encodes thepolypeptide. The outer membrane protein is a 63 kD protein originallyisolated from Leptospira alstoni which has been termed OmpL2 and is apathogen-associated exported protein of Leptospira. This immunogenicpolypeptide is useful in a pharmaceutical composition for inducing animmune response to pathogenic Leptospira.

[0017] The invention includes a method of producing the polypeptideportion of an outer membrane protein of Leptospira using recombinant DNAtechniques. The gene for the L. alstoni OmpL2 outer membrane protein iscloned into a plasmid vector which is then used to transform E. coli.When the OmpL2 gene is expressed in E. coli, the polypeptide producedhas a molecular weight of approximately 63 kD as determined bySDS-polyacrylamide gel electrophoresis.

[0018] Recently, one approach to studying genes encoding exportedleptospiral proteins was developed based on the concept underlyingTnphoA transposition (Boquet, et al., J. Bacteriol. 169:1663, 1987;Hoffman, et al., Proc. Natl. Acad. Sci. USA, 82:5107, 1985; Manoil, etal., Science 233:1403, 1986; Manoil, et al., J. Bacteriol. 172:515,1990). The system utilizes a phoA expression vector termed pMG, thatcontains an alkaline phosphatase (AP) gene lacking its signal sequence,together with the E. coli mutant strain KS330 (Strauch, et al., Proc.Natl. Acad. Sci., USA 85:1575, 1988), which possesses a leaky outermembrane, to identify genes encoding signal peptide export-dependentproteins which may function as virulence determinants. The screen forgenes which encode exported proteins is done by identifying blue-halocolonies. The utility of this system has been confirmed for bothTreponema pallidum (Blanco, et al., Mol. Microbiol. 5:2405, 1991) andLeptospira alstoni in which signal peptide containing proteins from bothorganisms were shown to be exported in E.coli. Such a method wasutilized for identification of the ompL2 gene of the invention.

[0019] Sequence analysis showed that the OmpL2 structural gene consistsof 1740 bases encoding a protein of 540 amino acids (SEQ ID NO:1 and 2).As expected for proteins to be exported beyond the inner membrane, thederived amino acid sequence begins with a 24-residue signal peptide. TheOmpL2 sequence contains 24 stretches of amphipathic beta-sheetstructure, consistent with outer membrane protein transmembranesegments, making it possible to propose a topological model with largesurface-exposed loops and short periplasmic loops typical of outermembrane proteins.

[0020] Comparison of the OmpL2 sequence with that of known outermembrane proteins revealed areas of homology to the TonB-dependent outermembrane proteins. The TonB-dependent proteins form ligand-specificchannels in the outer membrane of gram-negative bacteria. Sevenstretches of sequence have been found to be conserved in all TonB-dependent outer membrane proteins (Kadner, R. J., MolecularMicrobiology, 4:2027-2033, 1990). Sequence comparison, using the GAPprogram (Devereux, J., et al., Nucl. Acids Res., 12:387-395, 1984)demonstrated that the OmpL2 sequence is homologous in all seven of theconserved regions.

[0021] The bacterial genes for the OmpL2 outer membrane protein canlikely be derived from any strain of pathogenic Leptospira. Preferablythe protein is from Leptospira alstoni, strain RM52 (NationalLeptospirosis Reference Laboratory, Ames, Iowa). Leptospira alstoni isthe most current name for the pathogenic Leptospira previously groupedtogether in the family of Leptospira interrogans. The Leptospirainterrogans are publically available through the ATCC (Rockville, Md.),for example.

[0022] The invention provides polynucleotides encoding the LeptospiraOmpL2 protein. These polynucleotides include DNA and RNA sequences whichencode the protein. It is understood that all polynucleotides encodingall or a portion of OmpL2 are also included herein, so long as thesepolynucleotides exhibit the function of native or full length OmpL2,such as the ability to induce or bind antibody. Such polynucleotidesinclude both naturally occurring and intentionally manipulated, forexample, mutagenized polynucleotides.

[0023] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationprocedures which are well known in the art. These include, but are notlimited to: 1) hybridization of probes to genomic libraries to detectshared nucleotide sequences and 2) antibody screening of expressionlibraries to detect shared structural features.

[0024] Hybridization procedures are useful for the screening ofrecombinant clones by using labeled mixed synthetic oligonucleotideprobes where each probe is potentially the complete complement of aspecific DNA sequence in the hybridization sample which includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. By using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific DNA clone by the hybridization of the target DNA to that singleprobe in the mixture which is its complete complement (Wallace, et al.,Nucleic Acid Research, 9:879, 1981).

[0025] Alternatively, an expression library can be screened indirectlyfor OmpL2 peptides having at least one epitope using antibodies toOmpL2. Such antibodies can be either polyclonally or monoclonallyderived and used to detect expression product indicative of the presenceof OmpL2 DNA.

[0026] Generally, a lambda gt11 library is constructed and screenedimmunologically according to the method of Huynh, et al., (in DNACloning:A Practical Approach, D. M. Glover, ed., 1:49, 1985).

[0027] The development of specific DNA sequences encoding OmpL2 can alsobe obtained by: (1) isolation of a double-stranded DNA sequence from thegenomic DNA, and (2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest.

[0028] DNA sequences encoding OmpL2 can be expressed in vitro by DNAtransfer into a suitable host cell. “Recombinant host cells” or “hostcells” are cells in which a vector can be propagated and its DNAexpressed. The term also includes any progeny of the subject host cell.It is understood that not all progeny are identical to the parental cellsince there may be mutations that occur at replication. However, suchprogeny are included when the terms above are used.

[0029] The term “host cell” as used in the present invention is meant toinclude not only prokaryotes, but also, such eukaryotes as yeasts,filamentous fungi, as well as plant and animal cells. The term“prokaryote” is meant to include all bacteria which can be transformedwith the gene for the expression of the OmpL2 outer membrane protein ofLeptospira. Prokaryotic hosts may include Gram negative as well as Grampositive bacteria, such as E. coli, S. typhimurium, and Bacillussubtilis.

[0030] A recombinant DNA molecule coding for the OmpL2 protein can beused to transform a host using any of the techniques commonly known tothose of ordinary skill in the art. Especially preferred is the use of aplasmid containing the OmpL2 coding sequence for purposes of prokaryotictransformation. Where the host is prokaryotic, such as E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method by procedures well known in the art. Alternatively,MgCl₂ or RbCl can be used. Transformation can also be performed afterforming a protoplast of the host cell.

[0031] In the present invention, the OmpL2 sequences may be insertedinto a recombinant expression vector. The term “recombinant expressionvector” refers to a plasmid, virus or other vehicle known in the artthat has been manipulated by insertion or incorporation of OmpL2 geneticsequences. Such expression vectors contain a promotor sequence whichfacilitates the efficient transcription of the inserted genetic sequencein the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be cultured according to means known in the art toachieve optimal cell growth. Various shuttle vectors for the expressionof foreign genes in yeast have been reported (Heinemann, et al., Nature,340:205, 1989; Rose, et al., Gene, 60:237, 1987). Biologicallyfunctional DNA vectors capable of expression and replication in a hostare known in the art. Such vectors are used to incorporate DNA sequencesof the invention.

[0032] Methods for preparing fused, operably linked genes and expressingthem in bacteria are known and are shown, for example, in U.S. Pat. No.4,366,246 which is incorporated herein by reference. The geneticconstructs and methods described therein can be utilized for expressionof Leptospira OmpL2 in prokaryotic hosts.

[0033] Examples of promoters which can be used in the invention are: recA, trp, lac, tac, and bacteriophage lambda p_(R) or p_(L). Examples ofplasmids which can be used in the invention are listed in Maniatis, etal., (Molecular Cloning, Cold Spring Harbor Laboratories, 1982).

[0034] Antibodies provided in the present invention are immunoreactivewith OmpL2 protein. Antibody which consists essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations are provided. Monoclonalantibodies are made from antigen containing fragments of the protein bymethods well known in the art (Kohler, et al., Nature, 256:495, 1975;Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).

[0035] The term “antibody” as used in this invention includes intactmolecules as well as fragments thereof, such as Fab, F(ab′)₂, and Fvwhich are capable of binding the epitopic determinant. These antibodyfragments retain some ability to selectively bind with its antigen orreceptor and are defined as follows:

[0036] (1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

[0037] (2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

[0038] (3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction;

[0039] F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

[0040] (4) Fv, defined as a genetically engineered fragment containingthe variable region of the light chain and the variable region of theheavy chain expressed as two chains; and

[0041] (5) Single chain antibody (“SCA”), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

[0042] Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

[0043] As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

[0044] Antibodies which bind to the OmpL2 polypeptide of the inventioncan be prepared using an intact polypeptide or fragments containingsmall peptides of interest as the immunizing antigen. The polypeptide ora peptide of SEQ ID NO:2 used to immunize an animal can be derived fromtranslated cDNA or chemical synthesis which can be conjugated to acarrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is then used to immunize the animal (e.g., a mouse,a rat, or a rabbit).

[0045] If desired, polyclonal or monoclonal antibodies can be furtherpurified, for example, by binding to and elution from a matrix to whichthe polypeptide or a peptide to which the antibodies were raised isbound. Those of skill in the art will know of various techniques commonin the immunology arts for purification and/or concentration ofpolyclonal antibodies, as well as monoclonal antibodies (See forexample, Coligan, et al., Unit 9, Current Protocols in Immunology, WileyInterscience, 1991, incorporated by reference).

[0046] It is also possible to use the anti-idiotype technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

[0047] Minor modifications of OmpL2 primary amino acid sequence mayresult in proteins which have substantially equivalent function comparedto the OmpL2 protein described herein. Such modifications may bedeliberate, as by site-directed mutagenesis, or may be spontaneous. Allproteins produced by these modifications are included herein as long asOmpL2 function exists.

[0048] Modifications of OmpL2 primary amino acid sequence also includeconservative variations. The term “conservative variation” as usedherein denotes the replacement of an amino acid residue by another,biologically similar residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

[0049] Isolation and purification of microbially expressed protein, onfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

[0050] The invention extends to any host modified according to themethods described, or modified by any other methods, commonly known tothose of ordinary skill in the art, such as, for example, by transfer ofgenetic material using a lysogenic phage, and which result in aprokaryote expressing the Leptospira gene for OmpL2 protein. Prokaryotestransformed with the Leptospira gene encoding the OmpL2 protein areparticularly useful for the production of polypeptides which can be usedfor the immunization of an animal (e.g., a rabbit).

[0051] In one embodiment, the invention provides a pharmaceuticalcomposition useful for inducing an immune response to pathogenicLeptospira in an animal comprising an immunologically effective amountof OmpL2 in a pharmaceutically acceptable carrier. The term“immunogenically effective amount,” as used in describing the invention,is meant to denote that amount of Leptospira antigen which is necessaryto induce in an animal the production of an immune response toLeptospira. The OmpL2 outer membrane protein of the invention isparticularly useful in sensitizing the immune system of an animal suchthat, as one result, an immune response is produced which amelioratesthe effect of Leptospira infection.

[0052] The OmpL2 outer membrane protein can be administered parenterallyby injection, rapid infusion, nasopharyngeal absorption, dermalabsorption, and orally. Pharmaceutically acceptable carrier preparationsfor parenteral administration include sterile or aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Carriers forocclusive dressings can be used to increase skin permeability andenhance antigen absorption. Liquid dosage forms for oral administrationmay generally comprise a liposome solution containing the liquid dosageform. Suitable forms for suspending the liposomes include emulsions,suspensions, solutions, syrups, and elixirs containing inert diluentscommonly used in the art, such as purified water. Besides the inertdiluents, such compositions can also include adjuvants, wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

[0053] It is also possible for the antigenic preparations containing theOmpL2 protein of the invention to include an adjuvant. Adjuvants aresubstances that can be used to nonspecifically augment a specific immuneresponse. Normally, the adjuvant and the antigen are mixed prior topresentation to the immune system, or presented separately, but into thesame site of the animal being immunized. Adjuvants can be looselydivided into several groups based on their composition. These groupsinclude oil adjuvants (for example, Freund's Complete and Incomplete),mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica,alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, and carbon), polynucleotides (forexample, poly IC and poly AU acids), and certain natural substances (forexample, wax D from Mycobacterium tuberculosis, as well as substancesfound in Corynebacterium parvum, Bordetella pertussis, and members ofthe genus Brucella).

[0054] In another embodiment, a method of inducing an immune response topathogenic Leptospira in animal is provided. Many different techniquesexist for the timing of the immunizations when a multiple immunizationregimen is utilized. It is possible to use the antigenic preparation ofthe invention more than once to increase the levels and diversity ofexpression of the immune response of the immunized animal. Typically, ifmultiple immunizations are given, they will be spaced two to four weeksapart. Subjects in which an immune response to Leptospira is desirableinclude swine, cattle and humans.

[0055] Generally, the dosage of OmpL2 protein administered to an animalwill vary depending on such factors as age, condition, sex and extent ofdisease, if any, and other variables which can be adjusted by one ofordinary skill in the art.

[0056] The antigenic preparations of the invention can be administeredas either single or multiple dosages and can vary from about 10 ug toabout 1,000 ug for the Leptospira OmpL2 antigen per dose, morepreferably from about 50 ug to about 700 ug OmpL2 antigen per dose, mostpreferably from about 50 ug to about 300 ug OmpL2 antigen per dose.

[0057] When used for immunotherapy, the monoclonal antibodies of theinvention may be unlabeled or labeled with a therapeutic agent. Theseagents can be coupled either directly or indirectly to the monoclonalantibodies of the invention. One example of indirect coupling is by useof a spacer moiety. These spacer moieties, in turn, can be eitherinsoluble or soluble (Diener, et al., Science, 231:148, 1986) and can beselected to enable drug release from the monoclonal antibody molecule atthe target site. Examples of therapeutic agents which can be coupled tothe monoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

[0058] The labeled or unlabeled monoclonal antibodies of the inventioncan also be used in combination with therapeutic agents such as thosedescribed above. Especially preferred are therapeutic combinationscomprising the monoclonal antibody of the invention and immunomodulatorsand other biological response modifiers.

[0059] When the monoclonal antibody of the invention is used incombination with various therapeutic agents, such as those describedherein, the administration of the monoclonal antibody and thetherapeutic agent usually occurs substantially contemporaneously. Theterm “substantially contemporaneously” means that the monoclonalantibody and the therapeutic agent are administered reasonably closetogether with respect to time. Usually, it is preferred to administerthe therapeutic agent before the monoclonal antibody. For example, thetherapeutic agent can be administered 1 to 6 days before the monoclonalantibody. The administration of the therapeutic agent can be daily, orat any other interval, depending upon such factors, for example, as thenature of the disorder, the condition of the patient and half-life ofthe agent.

[0060] The dosage ranges for the administration of monoclonal antibodiesof the invention are those large enough to produce the desired effect inwhich the onset symptoms of the leptospiral disease are ameliorated. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the subject and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. Dosage can vary from about 0.1 mg/kg to about2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one ormore dose administrations daily, for one or several days. Generally,when the monoclonal antibodies of the invention are administeredconjugated with therapeutic agents, lower dosages, comparable to thoseused for in vivo diagnostic imaging, can be used.

[0061] The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

[0062] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

[0063] In a further embodiment, the invention provides a method ofdetecting a pathogenic Leptospira-associated disorder in a subjectcomprising contacting a cell component with a reagent which binds to thecell component. The cell component can be nucleic acid, such as DNA orRNA, or it can be protein. When the component is nucleic acid, thereagent is a nucleic acid probe or PCR primer. When the cell componentis protein, the reagent is an antibody probe. The probes are detectablylabeled, for example, with a radioisotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, a metal chelatoror an enzyme. Those of ordinary skill in the art will know of othersuitable labels for binding to the antibody, or will be able toascertain such, using routine experimentation.

[0064] For purposes of the invention, an antibody or nucleic acid probespecific for OmpL2 may be used to detect the presence of OmpL2polypeptide (using antibody) or polynucleotide (using nucleic acidprobe) in biological fluids or tissues. Any specimen containing adetectable amount of OmpL2 antigen or polynucleotide can be used. Apreferred specimen of this invention is blood, urine, cerebrospinalfluid, or tissue of endothelial origin.

[0065] When the cell component is nucleic acid, it may be necessary toamplify the nucleic acid prior to binding with a Leptospira specificprobe. Preferably, polymerase chain reaction (PCR) is used, however,other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used.

[0066] Another technique which may also result in greater sensitivityconsists of coupling antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, pyridoxal, and fluorescein, which canreact with specific antihapten antibodies.

[0067] Alternatively, OmpL2 polypeptide can be used to detect antibodiesto OmpL2 polypeptide in a specimen. The OmpL2 of the invention isparticularly suited for use in immunoassays in which it can be utilizedin liquid phase or bound to a solid phase carrier. In addition, OmpL2used in these assays can be detectably labeled in various ways.

[0068] Examples of immunoassays which can utilize the OmpL2 of theinvention are competitive and noncompetitive immunoassays in either adirect or indirect format. Examples of such immunoassays are theradioimmunoassay (RIA), the sandwich (immunometric assay) and theWestern blot assay. Detection of antibodies which bind to the OmpL2 ofthe invention can be done utilizing immunoassays which run in either theforward, reverse, or simultaneous modes, including immunohistochemicalassays on physiological samples. The concentration of OmpL2 which isused will vary depending on the type of immunoassay and nature of thedetectable label which is used. However, regardless of the type ofimmunoassay which is used, the concentration of OmpL2 utilized can bereadily determined by one of ordinary skill in the art using routineexperimentation.

[0069] The OmpL2 of the invention can be bound to many differentcarriers and used to detect the presence of antibody specificallyreactive with the polypeptide. Examples of well-known carriers includeglass, polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding OmpL2 or will be able to ascertain such, using routineexperimentation.

[0070] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, colloidal metals, fluorescent compounds, chemiluminescentcompounds, and bioluminescent compounds.

[0071] For purposes of the invention, the antibody which binds to OmpL2of the invention may be present in various biological fluids andtissues. Any sample containing a detectable amount of antibodies toOmpL2 can be used. Normally, a sample is a liquid such as urine, saliva,cerebrospinal fluid, blood, serum and the like, or a solid or semi-solidsuch as tissue, feces and the like.

[0072] The monoclonal antibodies of the invention, directed towardOmpL2, are also useful for the in vivo detection of antigen. Thedetectably labeled monoclonal antibody is given in a dose which isdiagnostically effective. The term “diagnostically effective” means thatthe amount of detectably labeled monoclonal antibody is administered insufficient quantity to enable detection of Leptospira OmpL2 antigen forwhich the monoclonal antibodies are specific.

[0073] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells, body fluid, or tissue having OmpL2 is detectable compared to thebackground. Further, it is desirable that the detectably labeledmonoclonal antibody be rapidly cleared from the circulatory system inorder to give the best target-to-background signal ratio.

[0074] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the subject. The dosage of monoclonal antibodycan vary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m²to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m².Such dosages may vary, for example, depending on whether multipleinjections are given, and other factors known to those of skill in theart.

[0075] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 key range, which may be readilydetected by conventional gamma cameras.

[0076] For in vivo diagnosis, radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

[0077] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0078] The monoclonal antibodies of the invention can be used to monitorthe course of amelioration of Leptospira associated disorder. Thus, bymeasuring the increase or decrease of Leptospira OmpL2 polypeptide orantibodies to OmpL2 polypeptide present in various body fluids ortissues, it would be possible to determine whether a particulartherapeutic regiment aimed at ameliorating the disorder is effective.

[0079] The materials for use in the method of the invention are ideallysuited for the preparation of a kit. Such a kit may comprise a carriermeans being compartmentalized to receive in close confinement one ormore container means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise a OmpL2binding reagent, such as an antibody. A second container may furthercomprise OmpL2 polypeptide. The constituents may be present in liquid orlyophilized form, as desired.

[0080] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

[0081] The following examples describe the identification of OmpL2 as animportant leptospiral outer membrane protein. The method by which theompL2 gene was cloned and sequenced is described. Sequence analysis andhomology studies are shown, further indicating that OmpL2 is an outermembrane protein of pathogenic Leptospira and therefore is an excellentvaccine candidate.

Example 1 Cloning of ompL2

[0082] The ompL2 gene was identified using an approach foridentification of genes encoding exported leptospiral proteins byscreening for blue-halo colonies using the pMG expresssion vector and E.coli KS330 (Blanco, et al., Molecular Microbiology, 5:2405, 1991;Giladi, et al., J. Bacteriol., 175:4129, 1993). The pMG vector is a phoAexpression vector, which, like TnphoA, is useful in identifying genesencoding membrane-spanning sequences or signal peptides. This cloningsystem has been modified to facilitate the distinction of outer membraneand periplasmic alkaline phosphatase (AP) fusion proteins from innermembrane AP fusion proteins by transforming pMG recombinants into E.coli KS330, the strain first used in the “blue halo” assay described byStrauch and Beckwith (Proc. Natl. Acad. Sci., USA 85:1576, 1988). Thelipoprotein mutation Ipp-5508 of KS330 results in an outer membrane thatis leaky to macromolecules, and its degP4 mutation greatly reducesperiplamic proteolytic degradation of AP fusion proteins. pMG AP fusionscontaining cleavable signal peptides, including the E. coli periplasmicprotein β-lactamase, OmpA and MOMP and Tp9, a Treponema palladum APrecombinant, have been shown to diffuse through the leaky outer membraneprotein of KS330 and result in blue colonies with blue halos (Giladi, etal., supra). In contrast, inner membrane AP fusions derived from E.coliproteins, including leader peptidase, SecY, and the tetracyclineresistance gene product, resulted in blue colonies without blue halos.The pMG/KS330r- cloning and screening approach identifies genes encodingproteins with cleavable signal peptides and therefore is useful in theidentification of genes encoding potential virulence factors.

[0083]Escherichia coli strains were grown at 37° C. on Luria-Bertanimedium. All restriction endonucleases and DNA-modifying enzymes wereused in accordance with the specifications of the manufacturer (BethesdaResearch Laboratories, Inc., Gaithersburg, Md., or Boehringer MannheimBiochemicals, Indianapolis, Ind.).

[0084]L. alstoni strain RM52 (National Leptospirosis ReferenceLaboratory, Ames, Iowa) genomic DNA was prepared by the method ofYelton, D. B., and N. W. Charon, (Gene, 28:147, 1984). Genomic DNA waspartially digested with Sau3A to a mean size of about 3.0 kb, ligated toBamHI-digested pMG and transformed into KS330r-. Approximately, 80,000recombinant clones were screened on XP-IPTG-containing plates (Giladi,et al., supra), and about 10,000 clones were screened on XP plateswithout IPTG, yielding 226 blue colonies. Clones producing blue colonieswere subcultured and spotted on high IPTG, high XP plates resulting inblue colonies, 66 of which showed blue halo formation. One such cloneshowing a blue halo, designated L2.086, was chosen for further study.This clone contained a 237 bp insert in pMG. The clone was identified asan outer membrane protein since it contained a leader sequence andleader peptidase I cleavage site (as determined from nucleic and deducedamino acid sequence) as indicated in FIG. 1 (↑).

[0085] The remainder of the ompL2 gene was cloned on 3.0 kb EcoRIfragment. A library of the DNA from L. alstoni was generated in the λZap II vector system (Stratagene, San Diego, Calif.). Followingdigestion with EcoRI, the DNA fragments were ligated into the phagevector. The library was packaged and plated according to themanufacturer's recommendations. Approximately 10,000 plaques wereplated, transferred to filters in duplicate, and processed as previouslydescribed (Maniatis, et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1982). An oligonucleotide probe based on theL2.086 insert was radiolabled as described (Maniatis, et al., supra) andused for plaque hybridizations. Positive recombinant pBluescript SK(−)clones were recovered by in vivo excision according to themanufacturer's instructions.

Example 2 Sequence Analysis for OmpL2

[0086] The L2.086 insert was sequenced in pMG by using thedideoxynucleotide chain termination method described by Sanger, et al.,(Proc. Natl. Acad. Sci. USA, 74:5463, 1977) and [α-³⁵S]-dATP (SeeGiladi, et al., supra). The remainder of the ompL2 gene was sequencedusing standard M13 primers and custom oligonucleotide primerssynthesized at UCLA, Dept. of Microbiology & Immunology for sequencingdouble-stranded templates. Sequencing reactions were performed for bothstrands using the Deaza T7 Sequencing kit protocol as described byPharmacia Biotech, Inc., and [α-³⁵S]dATP (specific activity, 1,000Ci/mmol). DNA and deduced amino acid sequences were analyzed using DNAStrider 1.0 (Marck, C., Nucl. Acids Res. 16:1829, 1988). Proteinhomology searches were performed with the Profilesearch and FASTAprograms found in the University of Wisconsin Genetics Computer Group(GCG), Inc., package, ver. 7.0 (Devereux, et al., Nucl. Acids Res.12:387, 1984).

[0087] An open reading frame of 1740 bp was identified, which wouldencode a 540-amino-acid polypeptide with a predicted molecular mass of63-kDa (FIG. 1). A Shine-Dalgarno ribosome binding site (RBS) wasidentified upstream from the ATG start codon, as well as putative −35and −10 promoter regions. The TAA stop codon is indicated by anasterisk. Data base searching using the FASTA and ProfileSearch programsfailed to reveal significant amino acid homologies. However, secondarystructure analysis predicted numerous areas of amphipathic beta-sheets,consistent with outer membrane protein transmembrane segments. Ofspecial note is the carboxy-terminal phenylalanine, a feature which ishighly conserved among outer membrane proteins (Struyve, M., et al., J.Mol. Biol., 218:141-148, 1991).

[0088] Comparison of the OmpL2 sequence with that of known outermembrane proteins revealed areas of homology to the TonB-dependent outermembrane proteins. The TonB-dependent proteins form ligand-specificchannels in the outer membrane of gram-negative bacteria. Sevenstretches of sequence have been found to be conserved in all TonB-dependent outer membrane proteins (Kadner, R. J., MolecularMicrobiology, 4:2027-2033, 1990). Sequence comparison, using the GAPprogram (Devereux, J., et al., Nucl. Acids Res., 12:387-395, 1984)demonstrated that the OmpL2 sequence is homologous in all seven of theconserved regions (FIG. 2). Peptide alignment between OmpL2 and eightTonB-dependent outer membrane proteins, for all seven regions ofhomology identified by Kadner, supra. Domain 1 is the “TonB box” whichhas been implicated in the direct interaction of Ton B with outermembrane receptors. OmpL2 is aligned with TBP1 (N. gonorrhoeaetransferrin-binding protein 1); BtuB (E. coli vitamin B₁₂ receptor); Cir(E. coli colicin I receptor); IutA (E. coli aerobactin receptor); FhuA(E. coli ferrichrome receptor); PupA (P. putida pseudobactin receptor);IrgA (V. cholerae iron-regulated outer membrane protein); FoxA (Y.enterocolitica ferrioxamine receptor). Asterisks mark positions ofcomplete identity in all nine proteins. Positions are indicated whereOmpL2 has a functionally similar amino acid as all (|), half (:), or 25%(.) of the other eight proteins, as predicted by the Mutation Matrix ofDayhoff. (in M. O. Dayhoff (ed.), Atlas of protein sequence andStructure, Vol. 5, Suppl. 3, National Biomedical Research Fdn.,Washington, D.C.).

[0089] The first of these segments is known as the TonB box, which ischaracterized by the following consensus sequence: Thr-X-Y-Val. TheOmpL2 TonB box retains the Threonine, but there is a conservativesubstitution of Isoleucine for Valine. A substitution at this positionis unprecidented among the known TonB-dependent outer membrane proteins,however, spirochetes occupy one of the deepest branches in eubacterialevolution and OmpL2 would be the first spirochetal TonB-dependent outermembrane protein to be identified. Mutagenesis studies demonstrate thatinteraction of TonB-dependent outer membrane proteins with TonB arehighly tolerant of amino acid substitutions within the TonB box, even atthe invariant Valine positions (Gudmundsdottir, A., et al., Journal ofBacteriology, 171:6526-6533, 1989).

Example 3 Topology of OmpL2

[0090] The topology of the E. coli TonB-dependent outer membraneprotein, FepA, has been studied using monoclonal antibodies and deletionmutagenesis (Rutz, J. M., et al., Science, 258:471-474, 1992). Atopology for the Y. enterocolica TonB-dependent outer membrane protein,FoxA, has also been proposed (Baumler, A. J., et al., MolecularMicrobiology, 6:1309-1321, 1992). The OmpL2 sequence contains 24stretches of amphipathic beta-sheets, consistent with transmembranesegments, making it possible to propose a topological model with largesurface-exposed loops and short periplasmic loops typical of outermembrane proteins (FIG. 3). The membrane-spanning beta-sheets are shownwithin rectangles in a staggered array with the hydrophobic,membrane-facing residues on the right side of the array.

Example 4 Expression of ompL2 During Iron Depletion

[0091] Studies show that OmpL2 is produced in greater amounts by L.alstoni when grown in iron-depleted media (bovuminar (Invirogen, N.Y.)containing 50 μM dipyridyl, an iron chelator). There is a potentialFur-binding site in the promoter region upstream of the ompL2 gene,which would also indicate that expression of ompL2 is turned on iniron-limiting conditions. This suggests that expression of OmpL2 occurswhen Leptospira are in the host, a feature common to most of the Ton-Bdependent outer membrane proteins. An outer membrane protein which isproduced by a bacterial pathogen when it enters the host would be anideal vaccine candidate.

Example 5 Southern and Northern Blot Analysis

[0092] Southern blot analysis is performed as described previously byManiatis, et al., supra. A probe from ompL2 is labeled at its 5′ endwith [γ-³²P]ATP (5,000 Ci/mmol; Amersham Corp., Arlington Heights, Ill.)and T4 polynucleotide kinase followed by purification over a BioSpin 6column (Bio-rad Laboratories, Hercules, Calif.). Membranes containingDNA from various Leptospira species are hybridized overnight at 37° C.with 1×10⁶ cpm/ml of hybridization buffer.

[0093] For Northern blot analysis, total cellular RNA is isolated fromL. alstoni by the method as previously described (Maniatis, et al.,supra). Approximately 15 μg of RNA is electrophoresed in duplicatethrough a 1.5% agarose-formaldehyde gel and transferred tonitrocellulose. The filters are probed with PCR-generated DNA fragmentsof ompL2 gene radiolabled with [α-³²P]dATP using the Random Primers DNALabeling System (BRL). Hybridizations are conducted as previouslydescribed (Maniatis, et al., supra).

Example 6 Cloning of the ompL2 Gene into the pRCET Expression Vector

[0094] The pBluescript plasmid containing the ompL2 gene was digestedwith HincII and Clal. The resulting DNA fragment encoding thecarboxy-terminal half of the OmpL2 protein was isolated by agarose gelelectrophoresis, and ligated into pRSET (Invitrogen, San Diego, Calif.)digested with PvuIII and Csp451. The resulting construct, pRSET-ompL2,encodes a fusion protein containing a 41 amino acid His6 binding site atthe amino terminus of OmpL2. The six histidines allow for pH-dependentaffinity purification of the fusion protein on a nickel resin column tothe exclusion of E. coli proteins. The pRSET fusion protein is under T7promoter control. After transformation of pRSET-ompL2 into E. coli DH5α,milligram quantities of the His6-OmpL2 fusion protein are produced inthe presence of isopropyl-β-D-thiogalactoside (IPTG, Sigma).

Example 7 Immunization of Rabbits with Purified OmpL2

[0095] The His6-OmpL2 fusion protein is separated from other insolublematerials by SDS-PAGE. The His6-OmpL2 band containing about 50micrograms of protein is cut out of the acrylamide gel, dessicated,ground to powder, mixed with Freund's complete adjuvant and inoculatedsubcutaneously and intramuscularly into a New Zealand White male rabbit.Additional His6-OmpL2 fusion protein is solubilized in 6M guanidine andpurified over the nickel resin column and dialyzed in 10 mM Tris, pH8.0. The secondary immunization is given six weeks after the primaryimmunization using roughly 50 micrograms of purified His6-OmpL2 fusionprotein in Freund's incomplete adjuvant. The rabbit is bled two weeksafter the secondary immunization. The post-boost antiserum will reactwith the 63-kDa antigen on immunoblots of whole L. alstoni separated bySDS-PAGE. Immunoblots of L. alstoni fractioned with TX-114 revealreactivity with the 63-kDa OmpL2 antigen in the whole organism anddetergent phase, but not the aqueous phase or insoluble pellet.

Example 8 Surface Localization with Immunoelectron Microscopy

[0096] Having obtained a highly specific immunological reagent forlocalization studies, preliminary immunoelectron microscopy experimentscan be conducted. A 20 μl suspension of 10⁷ L. alstoni is added to 0.5ml of heat-inactivated anti-OmpL2 antiserum or preimmune serum from thesame rabbit and incubated for one hour with mixing. The bacteria arefixed for 30 minutes by addition of 250 μl of 0.75% glutaraldehyde in100 mM cacodylate buffer, pH 7.0. The bacteria are washed, applied toelectron microscopy grids, and probed with protein G-colloidal gold (10nm particles).

Example 9 Expression of OmpL2 with the pTrc 99A Expression Vector

[0097] The His6 fusion protein is well suited for purification, but isnot appropriate for immunoblotting studies because of the potential forbackground reactivity to the 41 additional amino acids containing theHis6 binding site. Preimmune sera from one of the rabbits reacts withthe His6-OmpL2 fusion protein, but not with native OmpL2. A BgI II-HindII fragment is isolated from the pRCET-ompL2 vector by gelelectrophoresis and cloned into the pTrc99A expression vector(Pharmacia) which had been reading frame adjusted with a 10-mer Nco Ilinker. The pTtrc99A-ompL2 construct, transformed into E. coli DH5αexpresses the entire mature OmpL2 protein, plus a start methionine andonly five additional amino acids supplied by the vector. E. coli DH5αcontaining the original pTrc99A vector serves as a negative control.Bacterial proteins are separated by SDS-PAGE and transferred tonitrocellulose, and probed with antisera from rabbits immunized with avariety of pathogenic Leptospira strains (antisera supplied by Dr.Arnold Kaufmann, Centers for Disease Control, Atlanta). Reactivity toOmpL2 is likely demonstrated with antisera to L. interrogans, serovarsicterohaemorrhagiae, pomona, and bratislava, L. alstoni, serovarsgrippotyphosa and Mozdok, L. santarosai, serovars bakeri and canalzonae,and L. weilii, serovar celledoni. OmpL2 is likely not only expressed,but also antigenically conserved among pathogenic Leptospira, a featurethat would make it an excellent vaccine candidate.

[0098] The foregoing is meant to illustrate, but not to limit, the scopeof the invention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

Summary of Sequences

[0099] SEQ ID NO:1 is the nucleotide sequence and deduced amino acidsequence of ompL2.

[0100] SEQ ID NO:2 is the deduced amino acid sequence of OmpL2.

1. An isolated polypeptide comprising the amino acid sequence of OmpL2.2. The polypeptide of claim 1, wherein the polypeptide has a molecularweight of about 63 kD.
 3. The polypeptide of claim 1, wherein thepolypeptide has essentially the amino acid sequence of FIG.
 1. 4. Thepolypeptide of claim 1, wherein the OmpL2 is from Leptospira alstoni. 5.The polypeptide of claim 4, wherein the OmpL2 is from a serovar ofLeptospira alstoni selected the group consisting of grippotyphosa andMozdok.
 6. The polypeptide of claim 1, wherein the OmpL2 is fromLeptospira interrogans.
 7. The polypeptide of claim 6, wherein the OmpL2is from a serovar of Leptospira interrogans selected from the groupconsisting of icterohaemorrhagiae, pomona and bratislava.
 8. An isolatedpolynucleotide sequence which encodes the polypeptide of claim
 1. 9. Thepolynucleotide sequence of claim 8, wherein the polynucleotide is DNA.10. The polynucleotide of claim 8, wherein the ompL2 sequence isselected from the group consisting of a. the nucleotide sequence of FIG.1, wherein T can also be U; b. nucleic acid sequences complementary tothe nucleotide sequence of FIG. 1; and c. fragments of a. or b. that areat least 15 bases in length and which will selectively hybridize togenomic DNA which encodes the polypeptide of FIG.
 1. 11. Thepolynucleotide sequence of claim 8, wherein the polynucleotide is RNA.12. A recombinant expression vector containing the polynucleotide ofclaim
 8. 13. The expression vector of claim 12, wherein the vector is aplasmid.
 14. The vector of claim 12, wherein the polynucleotide sequenceis from L. alstoni.
 15. A host cell transformed with the expressionvector of claim
 12. 16. The host cell of claim 15, wherein the cell is aprokaryote.
 17. The prokaryote of claim 16, which is E. coli.
 18. Thehost cell of claim 15, wherein the cell is a eukaryote.
 19. A method ofproducing OmpL2 polypeptide which comprises: a. transforming a host withthe polynucleotide of claim 8; and b. expressing the polynucleotide inthe host.
 20. The method of claim 19, which further comprises isolatingthe OmpL2 polypeptide.
 21. The method of claim 19, wherein the host is aprokaryote.
 22. A pharmaceutical composition useful for inducing animmune response to pathogenic Leptospira in an animal comprising animmunogenically effective amount of OmpL2 in a pharmaceuticallyacceptable carrier.
 23. The pharmaceutical composition of claim 22,wherein the pharmaceutically acceptable carrier contains an adjuvant.24. A method of inducing an immune response to pathogenic Leptospira inan animal comprising immunizing the animal with the composition of claim22.
 25. A pharmaceutical composition useful for inducing an immuneresponse to pathogenic Leptospira in an animal comprising animmunogenically effective amount of antibody which binds OmpL2 in apharmaceutically acceptable carrier.
 26. An antibody which binds toOmpL2.
 27. The antibody of claim 26, wherein the antibody is polyclonal.28. The antibody of claim 26, wherein the antibody is monoclonal.
 29. Amethod of detecting a pathogenic Leptospira in a sample comprisingcontacting a pathogen-specific cell component in the sample with areagent which binds to the pathogen-specific cell component anddetecting the binding of the reagent to the cell component.
 30. Themethod of claim 29, wherein the pathogen-specific cell component isnucleic acid which encodes OmpL2 polypeptide.
 31. The method of claim30, wherein the nucleic acid is DNA.
 32. The method of claim 29, whereinthe nucleic acid is RNA.
 33. The method of claim 29, wherein thepathogen specific cell component is OmpL2 polypeptide.
 34. The method ofclaim 29, wherein the reagent is a probe.
 35. The method of claim 34,wherein the probe is nucleic acid.
 36. The method of claim 34, whereinthe probe is an antibody.
 37. The method of claim 36, wherein theantibody is polyclonal.
 38. The method of claim 36, wherein the antibodyis monoclonal.
 39. The method of claim 34, wherein the probe isdetectably labeled.
 40. The method of claim 39, wherein the label isselected from the group consisting of a radioisotope, a bioluminescentcompound, a chemiluminescent compound, a fluorescent compound, a metalchelate, or an enzyme.
 41. The method of claim 29, wherein the sample isfrom an animal selected from the group consisting of human, swine andcattle.
 42. A method for detecting antibody to OmpL2 polypeptide in asample which comprises contacting the sample with OmpL2 polypeptideunder conditions which allow the antibody to bind to OmpL2 polypeptideand detecting the binding of the antibody to the OmpL2 polypeptide. 43.The method of claim 42, wherein the OmpL2 polypeptide is detectablylabelled.
 44. A kit useful for the detection of OmpL2 polypeptide, thekit comprising carrier means being compartmentalized to receive in closeconfinement therein one or more containers comprising a first containercontaining a OmpL2 binding reagent.
 45. The kit of claim 44, wherein thereagent is an antibody.
 46. The kit of claim 45, wherein the antibody ishuman.
 47. The kit of claim 45, wherein the antibody is monoclonal. 48.A kit useful for the detection of OmpL2 polynucleotide, the kitcomprising carrier means being compartmentalized to receive in closeconfinement therein one or more containers comprising a first containercontaining a OmpL2 polynucleotide binding reagent.
 49. The kit of claim48, wherein the binding reagent is nucleic acid.
 50. A kit useful forthe detection of antibody to OmpL2 polypeptide, the kit comprisingcarrier means being compartmentalized to receive in close confinementtherein one or more containers comprising container containing OmpL2polypeptide.